Inositol in Disease and Development: Roles of Catabolism via myo-Inositol Oxygenase in Drosophila melanogaster

Inositol depletion has been associated with diabetes and related complications. Increased inositol catabolism, via myo-inositol oxygenase (MIOX), has been implicated in decreased renal function. This study demonstrates that the fruit fly Drosophila melanogaster catabolizes myo-inositol via MIOX. The levels of mRNA encoding MIOX and MIOX specific activity are increased when fruit flies are grown on a diet with inositol as the sole sugar. Inositol as the sole dietary sugar can support D. melanogaster survival, indicating that there is sufficient catabolism for basic energy requirements, allowing for adaptation to various environments. The elimination of MIOX activity, via a piggyBac WH-element inserted into the MIOX gene, results in developmental defects including pupal lethality and pharate flies without proboscises. In contrast, RNAi strains with reduced levels of mRNA encoding MIOX and reduced MIOX specific activity develop to become phenotypically wild-type-appearing adult flies. myo-Inositol levels in larval tissues are highest in the strain with this most extreme loss of myo-inositol catabolism. Larval tissues from the RNAi strains have inositol levels higher than wild-type larval tissues but lower levels than the piggyBac WH-element insertion strain. myo-Inositol supplementation of the diet further increases the myo-inositol levels in the larval tissues of all the strains, without any noticeable effects on development. Obesity and blood (hemolymph) glucose, two hallmarks of diabetes, were reduced in the RNAi strains and further reduced in the piggyBac WH-element insertion strain. Collectively, these data suggest that moderately increased myo-inositol levels do not cause developmental defects and directly correspond to reduced larval obesity and blood (hemolymph) glucose.

Inositol is a six-carbon sugar alcohol found in all eukaryotes and some prokaryotes. It can serve as a precursor of the membrane lipid phosphatidylinositol, act as a second messenger in signal transduction pathways, aid in osmoregulation, mediate endoplasmic reticulum stress (unfolded protein response), affect nucleic acid synthesis, or function as a

MIOX Homolog Identified in D. melanogaster (CG6910) Is Regulated in Response to Dietary myo-Inositol
To identify myo-inositol oxygenase (MIOX) in Drosophila melanogaster, BLASTP was performed using the 285 amino acid human MIOX protein sequence as a query. This revealed one candidate (CG6910) with~55% identity spanning the entire protein. Although there are two splice variants of CG6910, B and C, listed in Flybase [59], 5 RACE experiments previously performed in this lab did not detect the C variant in larvae or adults. Moreover, high-throughput expression data (RNA -Seq Signal by Region) and the G-browse visual display reveal that RNA transcripts unique to the C variant region are rare or undetectable in all stages of development and in all tissues examined [59]. To experimentally verify that CG6910 encoded the MIOX protein, three strains were obtained; two RNAi strains to reduce CG6910 transcript levels and one strain with a piggyBac WH-element inserted into CG6910 ( Figure 1A). RNAi strains to reduce CG6910 transcript levels and one strain with a piggyBac WH-element inserted into CG6910 ( Figure 1A). MIOX mRNA levels of larvae (left) and adults (right) grown on rich or semi-defined sucrose (CAA-S) or semi-defined myo-inositol (CAA-I) food. Normalized to RpL32, mean ± SE of three independent trials are represented. Control strains ActGal4-3/TbGFP and CyOGFP/+; ActGal4-3/+ were indistinguishable from the wild-type control Canton-S results. n.s. = not significant, * p < 0.05, ** p< 0.005, *** p < 0.0001 as indicated, determined by two-tailed t-test. (C) MIOX enzyme assays to determine myo-inositol oxygenase specific activity in crude lysates of larvae and adults (as indicated) grown on rich, CAA-S, or CAA-I food. Mean ± SE of three independent trials are represented. There was no detectable MIOX activity in crude lysates of the homozygous piggyBac WH-element insertion strain (P-miox f01770 /P-miox f01770 ). Control strains ActGal4-3/TbGFP and CyOGFP/+: ActGal4-3/+ were indistinguishable from the wild-type control Canton-S results shown. * p < 0.05, *** p < 0.0001 as indicated, determined by two-tailed t-test.
To determine if the levels of CG6910 (MIOX) mRNA are regulated in response to myo-inositol, qRT-PCR experiments were performed. RNA was extracted from third-instar larvae and adults of five D. melanogaster strains. Two of the strains have different MIOX (CG6910) RNAi constructs with UAS GAL4 sequences. Both are controlled by GAL4 driven by the Actin 5C (Act5C) promoter (MIOXi2/+; +/Act5CGal4-3 and MIOXi3/Act5CGal4-3). Three control strains were also used (CS, ActGal4-3/Tb, and CyOGFP/+; ActGal4-3/Tb). The adults had slightly lower MIOX mRNA levels than the larvae. MIOX (CG6910) mRNA levels in MIOXi2/+; +/Act5Cgal4-3 and MIOXi3/Act5CGal4-3 were significantly lower than in the control strains. When larvae were grown on semi-defined food with myo-inositol as the sole sugar (CAA-I), the level of MIOX mRNA was significantly higher than when grown on semi-defined food with sucrose as the sole sugar (CAA-S) ( Figure 1B).
To examine if dietary myo-inositol affected MIOX specific activity, the conversion of myo-inositol to glucuronic acid by crude lysates of third-instar larvae and adults was monitored in the strains described above. There was slightly less activity detectable in the adults than the larvae. MIOX specific activity in larvae was significantly higher for all the strains when grown on CAA-I. Moreover, the specific activity of MIOX in crude lysates of MIOXi2/+, +/Act5CGal4-3, and MIOXi3/Act5CGal4-3 larvae was significantly lower than that of the control larvae ( Figure 1C). Even more striking is that there was no detectable MIOX activity in crude lysates of the homozygous piggyBac WH-element insertion strain (P-miox f01770 /P-miox f01770 ).

Dietary myo-Inositol Supports Survival of Wild-Type (CS) D. melanogaster Adults but Not of MIOX Knockdown Strains
To determine if MIOX plays a role in fruit fly survival, pupae of MIOXi2/+; +/Act5CGal4-3, MIOXi3/Act5CGal4-3, and the wild-type control strain (CS) were transferred to tubes with semi-defined food containing sucrose (CAA-S) or myo-inositol as the sole sugar (CAA-I), or no sugar (CAA-0). It was exciting to note that wild-type (CS) adult flies survived equally well on semi-defined food with either myo-inositol or sucrose as the sole sugar, demonstrating that there is sufficient myo-inositol catabolism to support survival of D. melanogaster ( Figure 2A).

Disruption of myo-Inositol Catabolism via Piggybac WH-Element Insertion in MIOX Results in Developmental Defects
The survival experiments described above did not include adult P-miox f01770 /P-miox f01770 flies, because these homozygotes are not viable as adults. As displayed in Figure  3A, ~16% of the homozygous P-miox f01770 /P-miox f01770 and ~76% of the control embryos developed to the pupal stage. Even more striking is that only ~6% of the homozygous Pmiox f01770 /P-miox f01770 pupae eclosed as adults, with most dying as pharate adults (not eclosing from the pupal case), in contrast to ~96% of the control strain eclosing as adults. The few P-miox f01770 /P-miox f01770 adults that eclosed from the pupal case died within two days and exhibited severe head morphological defects, most noticeably the lack of a proboscis ( Figure 3B). To confirm that the piggyBac WH-element in P-miox f01770 caused the pupal lethality and morphological defect, heterozygous strains with the element excised were generated. All three independently obtained excision lines (eleven stocks), with TM6, Tb balancer chromosomes, produced viable homozygous progeny in the expected ratio. Of the

Disruption of myo-Inositol Catabolism via Piggybac WH-Element Insertion in MIOX Results in Developmental Defects
The survival experiments described above did not include adult P-miox f01770 /Pmiox f01770 flies, because these homozygotes are not viable as adults. As displayed in Figure 3A,~16% of the homozygous P-miox f01770 /P-miox f01770 and~76% of the control embryos developed to the pupal stage. Even more striking is that only~6% of the homozygous P-miox f01770 /P-miox f01770 pupae eclosed as adults, with most dying as pharate adults (not eclosing from the pupal case), in contrast to~96% of the control strain eclosing as adults. The few P-miox f01770 /P-miox f01770 adults that eclosed from the pupal case died within two days and exhibited severe head morphological defects, most noticeably the lack of a proboscis ( Figure 3B). To confirm that the piggyBac WH-element in P-miox f01770 caused the pupal lethality and morphological defect, heterozygous strains with the element excised were generated. All three independently obtained excision lines (eleven stocks), with TM6, Tb balancer chromosomes, produced viable homozygous progeny in the expected ratio. Of the 998 pupae examined (350 homozygotes; 648 heterozygotes (Tb)), the same percentage of homozygotes and heterozygotes eclosed. Neither pupal lethality nor morphological defects were observed in the homozygous excision progeny. The excision of the element reverted the phenotype. 998 pupae examined (350 homozygotes; 648 heterozygotes (Tb)), the same percentage of homozygotes and heterozygotes eclosed. Neither pupal lethality nor morphological defects were observed in the homozygous excision progeny. The excision of the element reverted the phenotype.

Reduced myo-Inositol Catabolism Increases myo-Inositol Levels in Larvae
To assess if the developmental defect observed in the P-miox f01770 /P-miox f01770 strain was due to reduced catabolism yielding increased myo-inositol levels, assays were performed using carcasses of third-instar larvae grown on rich food with 0 or 50 µ M myoinositol supplementation. Higher myo-inositol levels were observed in the tissues of larvae with decreased myo-inositol catabolism ( Figure 4A). The highest level of myo-inositol is apparent in the P-miox f01770 /P-miox f01770 larval tissues which had no detectable myo-inositol catabolic activity via MIOX. In all the strains, myo-inositol levels increased when the standard rich food was supplemented with 50 µ M of myo-inositol ( Figure 4A).

Increased myo-Inositol Decreases Larval Obesity and Hemolymph Glucose
To examine the relationship between MIOX and the diabetic hallmarks, obesity and high hemolymph glucose, third-instar larvae grown on standard rich food with 0 or 50 µ M myo-inositol supplementation were assayed. Buoyancy, TAG, and glucose assays revealed that P-miox f01770 /P-miox f01770 larvae were the least obese with the lowest levels of On the left is the control heterozygous P-miox f01770 /TbGFP (N = 15) (indistinguishable from the wild-type control Canton-S), and on the right is P-miox f01770 /P-miox f01770 (N = 16). The arrows indicate the proboscis in the wild-type or the region lacking the proboscis in the piggyBac WH-element insertion strain.

Reduced myo-Inositol Catabolism Increases myo-Inositol Levels in Larvae
To assess if the developmental defect observed in the P-miox f01770 /P-miox f01770 strain was due to reduced catabolism yielding increased myo-inositol levels, assays were performed using carcasses of third-instar larvae grown on rich food with 0 or 50 µM myoinositol supplementation. Higher myo-inositol levels were observed in the tissues of larvae with decreased myo-inositol catabolism ( Figure 4A). The highest level of myo-inositol is apparent in the P-miox f01770 /P-miox f01770 larval tissues which had no detectable myo-inositol catabolic activity via MIOX. In all the strains, myo-inositol levels increased when the standard rich food was supplemented with 50 µM of myo-inositol ( Figure 4A).

Increased myo-Inositol Decreases Larval Obesity and Hemolymph Glucose
To examine the relationship between MIOX and the diabetic hallmarks, obesity and high hemolymph glucose, third-instar larvae grown on standard rich food with 0 or 50 µM myo-inositol supplementation were assayed. Buoyancy, TAG, and glucose assays revealed that P-miox f01770 /P-miox f01770 larvae were the least obese with the lowest levels of TAG and hemolymph glucose. In these assays, the two RNAi knockdown strains, MIOXi2/+; +/Act5CGal4-3 and MIOXi3/Act5CGal4-3, had intermediate levels relative to the control and the P-miox f01770 /P-miox f01770 strains ( Figure 4B-D). Dietary myo-inositol supplementation (50 µM) further reduced the proportion of obese larvae, TAG, and hemolymph glucose in all strains ( Figure 4B-D).

Discussion
This study examines the roles of myo-inositol catabolism using the model organism D. melanogaster. CG6910 was identified as the myo-inositol catabolic gene encoding myo-inositol oxygenase (MIOX), which is more than 55% identical (>70% similar) to human MIOX. The high level of identity among these two evolutionarily distant organisms demonstrates the conservation of MIOX structures in eukaryotes. There are two splice variants, B and C, listed in Flybase [59]; however, multiple experiments suggest that RNA transcripts unique to the C variant region are rare or undetectable in all stages of development and in all tissues examined [59]. If the C variant exists in larvae and adults, it comprises a small proportion of the MIOX transcripts and protein. Developmental proteome experiments reveal high levels of MIOX expression in late third-instar larvae (wandering and prepupae) and adults [60]. Temporal microarray and RNAseq data have shown the peak expression of MIOX (CG6910) mRNA in late third-instar larvae and adults [61]; therefore, an emphasis has been placed on examining third-instar larvae and adults.
This appears to be the first report to examine MIOX (CG6910) mRNA levels in animals, D. melanogaster larvae and adults, in response to dietary myo-inositol ( Figure 1B). A significantly higher level of MIOX mRNA is apparent in qRT-PCR experiments examining larvae fed myo-inositol as the sole sugar (CAA-I) compared to larvae fed sucrose as the sole sugar (CAA-S). MIOX mRNA levels were reduced in larvae and adults via two different RNAi constructs (MIOXi2/+; +/Act5CGal4-3 and MIOXi3/Act5CGal4-3) ( Figure 1B). Similar to the wild-type control, these strains have higher levels of MIOX mRNA when grown on CAA-I than when grown on CAA-S ( Figure 1B). Increased myo-inositol catabolism, via MIOX, has been implicated in decreased renal function [62]. Decreased MIOX mRNA levels via siRNA in transgenic mice expressing high levels of MIOX mRNA has been shown to reduce renal damage and associated endoplasmic reticulum stressors [63]. Yet, in this study using D. melanogaster, neither the RNAi strain nor the wild-type controls exhibited any gross morphological abnormalities, even with increased levels of MIOX mRNA when fed CAA-I.
An assay to measure MIOX-specific activity in D. melanogaster was established based on previously existing protocols for rat kidneys, hog kidneys, and fungi [28,31,32] (Figure 1C). Similar to the mRNA levels, MIOX-specific activity levels in MIOXi2/+; +/Act5CGal4-3 and MIOXi3/Act5CGal4-3 larvae and adults were lower than the control (CS) strain. Moreover, all the strains had increased levels of MIOX activity when fed CAA-I. Homozygous CG6910-MIOX (P-miox f01770 /P-miox f01770 ) larvae with a 7.2 kb piggyBac WH-element insertion disrupting the MIOX gene had no detectable MIOX activity. The MIOX-specific activity in crude lysates of control D. melanogaster larvae is 5.2 µmol/30 mins/mg, slightly more than that observed in adults, much more than that in rat kidney [29], and similar to that in the fungus Cryptococcus neoformans [32].
Survival experiments revealed that adult D. melanogaster have sufficient myo-inositol catabolism and transport to remain viable on semi-defined food with myo-inositol as the sole sugar/energy source (CAA-I) (Figure 2). The statistically significant results of six independent trials also showed that a reduction in MIOX expression via RNAi diminishes viability on CAA-I, mimicking survival on food with no sugar. The identical loss of the viability of both fly strains, MIOXi2/+; +/Act5CGal4-3 and MIOXi3/Act5CGal4-3, is particularly compelling, because the RNAi constructs are located on separate chromosomes and the strains were generated from two separate Vienna Drosophila Resource Center (VDRC) libraries (KK (phiC31) and GD (P-element), respectively). These libraries used different vectors and cloning methods and have been shown to have different off-target effects [64][65][66][67]. Since the two strains appeared phenotypically identical, the observed phenotypes in this study should be due to the decreased MIOX mRNA levels. Moreover, these results indicate that MIOX is a component of the primary myo-inositol catabolic pathway in D. melanogaster.
Adult P-miox f01770 /P-miox f01770 were not included in experiments because they are rarely viable, with only~6% of the pupae eclosing and the flies that eclose dying within two days ( Figure 3A). These results are similar to previously published findings that highly upregulated myo-inositol synthesis reduces eclosion to~9% [17]. As myo-inositol is a precursor of the phosphatidylinositol phosphates (PIPs), it is interesting to note that inositol phosphate kinase 2 (Ipk2) deletions and dysregulation of the expression of the phosphatidylinositol synthase gene (Pis) also cause pupal lethality [68,69]. Among the few P-miox f01770 /P-miox f01770 adults, the most jarring morphological defect is the lack of proboscises ( Figure 3B). This phenotype has been previously described when myo-inositol synthesis was highly upregulated [17]. The MIOX RNAi knockdown strains with intermediate levels of of myo-inositol in larval tissues did not display morphological abnormalities, paralleling previously published findings that lower but still elevated levels of myo-inositol synthesis did not produce the developmental defect [17]. Together, these data suggest that increased myo-inositol, not the process of synthesis or catabolism, contributes to or causes developmental defects. Deformities of fruit fly head structures have been observed with mutations disrupting the head involution defective (hid) [70,71] or the decapentaplegic (dpp) genes [72][73][74]. The morphological abnormalities observed in this study, however, seem to be unique to flies with elevated myo-inositol levels.
As could be predicted, less myo-inositol catabolism results in more myo-inositol in larval tissues, with the highest myo-inositol level observed in the strain with the lowest level of catabolism (P-miox f01770 /P-miox f01770 ). Not surprisingly, the intermediate levels of myo-inositol catabolism in the two RNAi knockdown strains MIOXi2/+; +/Act5CGal4-3 and MIOXi3/Act5CGal4-3 showed intermediate levels of myo-inositol in larval tissues ( Figure 4A). When rich food was supplemented with 50 µM of myo-inositol, the myoinositol levels in all the strains increased ( Figure 4A). Interestingly, whole MIOXi2/+; +/Act5CGal4-3 and MIOXi3/Act5CGal4-3 larvae fed rich food with 50 µM myo-inositol supplementation had more total myo-inositol than P-miox f01770 /P-miox f01770 larvae fed rich food without myo-inositol supplementation, yet only P-miox f01770 /P-miox f01770 displayed morphological defects ( Figure 3B). Larvae with hemolymph removed (carcasses) of MIOXi2/+; +/Act5CGal4-3 and MIOXi3/Act5CGal4-3 fed rich food with 50 µM myo-inositol supplementation had lower total myo-inositol than carcasses of P-miox f01770 /P-miox f01770 larvae fed rich food without myo-inositol supplementation ( Figure 4A). Apparently, adding dietary myo-inositol, and by doing so increasing the circulating myo-inositol levels, does not affect development but does further reduce obesity (buoyancy and TAG) and hemolymph glucose levels in all the strains ( Figure 4B,C). It is tantalizing to speculate that at least some of the developmental defects observed in P-miox f01770 /P-miox f01770 stem from abnormally high myo-inositol levels during embryogenesis prior to the organism feeding. Notably, abnormally high myo-inositol levels contribute to the pathology of some human disorders of neurological development and dysfunction [6,7,[75][76][77].
Alterations of myo-inositol metabolism been implicated in many human diseases and disorders including diabetes, obesity, and hyperglycemia. Low MIOX expression/activity, which should elevate myo-inositol levels, rescued mice and rats from renal injury and oxidative stress [27,62]. In this study, low MIOX levels were shown to reduce obesity and hyperglycemia in populations of D. melanogaster larvae. In addition, the supplementation of the rich food with 50 µM of myo-inositol further reduced obesity and hyperglycemia. In humans, dietary myo-inositol augmentation may mitigate obesity and hyperglycemia (high blood glucose) [4,7,78]. These results complement studies which established that reduction in the inositol 1,4,5-trisphosphate receptor (InsP3R), either by knockdown or mutation, resulted in obese adult fruit flies [79]. Moreover, these results are in agreement with studies demonstrating that an increase in myo-inositol, via the overexpression of the myo-inositol synthetic gene or the addition of dietary myo-inositol, decreased obesity and hyperglycemia in D. melanogaster [17]. In summary, increased myo-inositol, regardless of its source, can mitigate diabetes-associated obesity and hyperglycemia. This study, at the junction of metabolism and development, furthers the understanding of the importance of myo-inositol catabolism and the regulation of intracellular myo-inositol levels and may have implications for the treatment of diabetes and developmental disorders.  Austria). The RNAi strain MIOXi2/MIOXi2 is homozygous for an RNAi construct complementary to the third exon of CG6910 that was inserted via P-element to chromosome 2. The other RNAi strain, MIOXi3/Tb, also containing sequences complementary to the third exon of CG6910, is heterozygous for a different RNAi construct that was inserted via a phiC31 sequence to chromosome 3. Both RNAi constructs contain UAS GAL4 sequences controlled by GAL4 expression. MIOXi2/+; +/ActGal4-3 and MIOXi3/ActGal4-3 were generated by mating strains marked with GFP on corresponding balancer chromosomes. The third strain was generated by introducing a GFP-marked balancer third chromosome (TM6) into the P-miox f01770 /Tb, an existing strain with an approximately 7.2kb piggyBac WHelement inserted into the second intron of CG6910, and non-GFP non-tubby homozygotes (P-miox f01770 / P-miox f01770 ) were then readily identified. The piggyBac-element insertion in CG6910 was remapped and its location confirmed using flanking sequence data [80]. To create a double-marked (Tb/Sb) transposase strain, the strain harboring the transposase (#8283) was crossed to a third chromosome double balancer strain (#2537) introducing the Tb marked TM6B chromosome. These Tb marked flies were again crossed to the third chromo- To excise the piggyBac WH-element inserted in the MIOX gene (CG6910), the w[+]; piggyBac transposase; Tb/Sb strain was crossed to P-miox f01770 /Tb (#18471). According to Thibault et al. 2004 [81], excisions of this piggyBac WH-element are precise. Seventy-two single Tubby (Tb) progeny with dark red eyes (double w[+mC]) with curly wings (CyO) harboring both the transposase and the piggyBac WH-element were individually mated with w[*]; Tb/Sb. Eight of the seventy-two crosses produced white-eyed, straight-winged progeny, carrying neither the piggyBac WH-element (chromosome 3, CG6910) nor the transposase (chromosome 2, CyO, P{w[+mC] = FRT(w[+])Tub-PBac\T}2). Of these eight, three produced multiple white-eyed, straight-winged progeny which were used to establish stocks. Three or four individuals from each of the three independent lines (eleven flies) with putative piggyBac WH excisions (from the MIOX gene (CG6910)) were individually crossed to w[*]; Tb/Sb, and Tb progeny were selected.

Fly Stocks and Maintenance
Flies were maintained in standard laboratory conditions at 25 • C and 70-80% humidity on a 12 h:12 h light-dark cycle. All fly stocks were grown on either rich food (BDSC cornmeal food, https://bdsc.indiana.edu/information/recipes/bloomfood.html (accessed on 29 April 2011)) or modified food (per liter, 10 g of agar (Fisher Scientific, Waltham, MA, USA), 80 g of brewer's yeast (Genesee), 20 g of yeast extract (Fisher Scientific), 20 g of peptone (Fisher Scientific), and sucrose (Fisher Scientific) as indicated, [49]) with or without 50 µM of myo-inositol (Sigma, St. Louis, MO, USA) as indicated, which is sufficient to support growth of a homozygous Inos deletion mutant (inos ∆DF /inos ∆DF ) [16]. Semidefined food (casamino acids sucrose (CAA-S) and casamino acids myo-inositol (CAA-I)) was prepared essentially as described [82] with modifications [83]. Briefly, defined food contained 0.4 g of lecithin, 0.613 g of vitamin mix, 5.5 g of casamino acids, 3.15 g of agar, and 7.5 g of sugar (sucrose or myo-inositol) or no sugar per 100 mL. The vitamin mix was composed of 3 g of cholesterol, 0.02 g of thiamine, 0.01 g of riboflavin, 0.12 g of nicotinic acid, 0.16 g of calcium pantothenate, 0.25 g of pyridoxine, 0.016 g of biotin, 0.03 g of folic acid, 14 g of NaHCO 3 , 18.3 g of KH 2 PO 4 , 18.9 g of K 2 HPO 4 , and 6.2 g of MgSO 4 . Then, 350 µL of 30% Tegosept was added to the 100 mL of food. The results were normalized to the transcript levels of Drosophila melanogaster ribosomal protein L32 (RpL32). The following primers were used: MIOX exon 2-3 forward GACACCACCGATCCTCTAAAGG and reverse GGAAGGCGTGGATGATGT, RpL32 forward CCAGCATACAGGCCCAAGAT and reverse GCACTCTGTTGTCGATACCC.

Protein Extraction and the MIOX Activity Assay
The MIOX activity assays were established for D. melanogaster based on protocols for hog kidneys and fungi [31,32]. For each sample, ten flies or ten larvae were homogenized in 300 µL of 20 mM sodium acetate (pH 6.0), 2 mM L-cysteine (pH 4.5), 1 mM glutathione (pH 3.5), 1mM ferrous ammonium sulfate (pH 4.5), and 10 µL of protease inhibitor (Halt™ Protease Inhibitor Cocktail (100X), Thermo Scientific). Protein concentrations of the crude lysate cleared supernatants were determined using the Bradford Assay [85] with bovine serum albumin (Pierce™ Bovine Serum Albumin Standard, Thermo Scientific) and using a dye concentrate (5000006, Bio-Rad, Hercules, CA, USA) for the colorimetric analysis. myo-Inositol catabolism was assayed in 450 µL of 50 mM sodium acetate (pH 6.0), 2 mM L-cysteine (pH 3.5), 1 mM ferrous ammonium sulfate (pH 4.5), and 60 mM myo-inositol (or water) with 15 µg of crude lysate protein. The reactions were incubated at 30 • C. Then, 200 µL aliquots were removed at 0 and 30 min, immediately added to 55 µL of 30% trichloroacetic acid (TCA), and incubated at 100 • C for two minutes. The glucuronic acid concentrations of the cleared supernatants were determined by adding 500 µL of orcinol reagent (0.08 g of orcinol, 0.018 g of FeCl 3 and 20 mL of concentrated HCl) to 250 µL of the sample and were measured at 660 nm [28].

Survival Studies
Twenty CS or Act-Gal4-3/TbGFP virgin females and ten CS or MIOXi2/MIOXi2 or MIOXi3/TbGFP males were mated on rich food. For each of the six independent trials, twenty pupae from each cross were transferred onto rich food with 0 or 50 µM of myoinositol added, CAA-S, CAA-I, or CAA-0 (no sugar added). Survival was monitored daily.

Pupariation and Eclosion
Female and male adults (2:1) were placed in vials of standard rich food in a 25 • C incubator at 70-80% humidity on a 12 h:12 h light-dark cycle. The progeny (embryos) were sorted using the GFP marker and reconfirmed as larvae. To allow sufficient time for genotypes with developmental delays to eclose, the vials were checked daily for up to 28 days. The number of pupae were recorded, as was the number of adults that eclosed.

Light Microscopy
Fifteen control CS and sixteen independent experimental P-miox f01770 /P-miox f01770 specimens were viewed on a Nikon SMZ1500 microscope, and images were captured using a Micropublisher 6 color CCD camera system (Teledyne Q imaging, Surrey, BC, Canada).

myo-Inositol Assay
Five third-instar larval carcasses per sample were collected by puncturing five larvae and draining the hemolymph via centrifugation. The samples were homogenized in dH 2 O, and the Megazyme myoinositol assay kit (K-INOSL) was used as per the manufacturer's instructions. Three independent trials were performed.

Buoyancy Assay
Experiments were conducted essentially as described by Reis [48,54] using 20-30 3rdinstar larvae per sample, with initial results confirmed by the method of Hazegh and Reis [54]. A relationship between the percentage of larvae floating in a buoyancy assay and the percent body fat of the larvae has been established [48,86]. In this study, the relative obesity of a population of larvae is defined as the proportion of the larvae floating in the buoyancy assay.

Triacylglyceride (TAG) Assay
Experiments were performed essentially as previously described [39] and normalized to total protein (using the Bradford Assay described above). Six third-instar larvae per sample were homogenized in 1xPBS with 9.1% Tween, and the Serum Triglyceride Determination Kit (TR0100, Sigma, St. Louis, MO, USA) and Triglyceride Reagent (T2449, Sigma) were used as per the manufacturer's instructions for three independent trials.

Hemolymph Glucose Assay
Experiments were performed essentially as described by Tennessen et al. [39]. Hemolymph was collected by puncturing five third-instar larvae per replicate, and the Sigma Glucose (GO) Assay Kit GAGO-20 was used. Three independent trials were performed.

Computational Analyses
The National Center for Biotechnology Information's (NCBI) Basic Local Alignment Search Tool (BLASTP) with default settings (BLOSSUM 62) was used to identify the MIOX homolog in D. melanogaster.

Statistical Analyses
Standard error was calculated for all experiments. Mantel-Cox (log rank) tests were performed to calculate significance in survival studies. The p-values of pairwise comparisons were determined using Student's two-tailed t-test.

Conclusions
In the model organism Drosophila melanogaster, the elimination of myo-inositol catabolism and the associated high levels of myo-inositol cause severe developmental defects. A reduction in myo-inositol catabolism or dietary myo-inositol supplementation yields the beneficial effects of higher myo-inositol levels (reduced obesity and hemolymph (blood) glucose in Drosophila melanogaster third-instar larvae) without causing developmental defects. This suggests that dietary inositol supplementation may serve as a therapeutic agent.